1. Field of the Invention
This invention relates to methods and apparatus for detecting, characterizing and quantifying particulate matter suspended in a fluid. More specifically, the invention provides an integrated, affinity-binding based analytical system for detecting particulates, particularly cells, suspended in a fluid, especially a biological fluid. In particular, the invention provides a platform for performing an affinity-binding based assay for specifically binding particulates including cells, and a detection means for detecting the particulates specifically bound to a defined surface or chamber comprising the platform. In addition, the invention provides such analytical systems to facilitate cell accumulation in a specific cell accumulation area or chamber of the platform, allowing particulate counting and characterization using the platform, as well as high throughput screening of test compounds to determine the capacity of the compound to affect cell viability, metabolism or physiology. Devices for manipulating the platforms of the invention are provided comprising detection means operatively arranged relative to the platform, as well as devices that provide detecting means for manually-manipulated platforms. Methods for using the platforms of the invention are also provided.
2. Background of the Related Art
Determining the type, concentration and properties of particulates in a fluid is important in a variety of contexts. Dust and dirt particles in water, oil or other industrial fluids can negatively impact on the performance and useful lifetime of complex machinery. Pyrogens, including bacterial cells, in pharmaceutical products, or manufacturing facilities making such products, can compromise the safety and reliability of available drugs. Similarly, cells, particularly bacterial cells, that are themselves disease-causing (such as Salmonella spp.) or that make toxins (such as botulism toxin) are hazardous, and advantageously are screened in manufacturing and other settings where foodstuffs or other consumables are produced. Finally, mammalian cells, including sperm cells and hematopoietic cells, are usefully analyzed in the corresponding biological fluids for diagnostic and treatment monitoring purposes.
Certain methods and apparatus for detecting biological molecules and cells are known in the prior art.
U.S. Pat. No. 3,615,222 issued Oct. 26, 1971 to Mead discloses a specific binding method for detecting a component of a biological fluid.
U.S. Pat. No. 3,743,482 issued Jul. 3, 1973 to Eisentraut discloses a method for determining thyroid function.
U.S. Pat. No. 3,907,502 issued Sep. 23, 1975 to Brink discloses a method for identifying Bence Jones proteins.
U.S. Pat. No. 4,546,460 issued Oct. 8, 1985 to Ando discloses a videodisc autofocus device.
U.S. Pat. No. 5,009,997 issued Apr. 23, 1991 to Shah et al. discloses two-site immunometric sandwich assay.
U.S. Pat. No. 5,091,318 issued Feb. 25, 1992 to Anawis et al. discloses binding of allergens to a solid phase.
International Application, Publication No. WO92/07243, published on Apr. 30, 1992 in the name of Cellpro, disclose the use of a biological particle separator.
U.S. Pat. No. 5,137,031 issued Aug. 11, 1992 to Guirguis discloses a urine testing apparatus and cell collection.
U.S. Pat. No. 5,278,048 issued Jan. 11, 1994 to Parce et al. discloses an apparatus for detecting the effect of a test compound on a living cell.
U.S. Pat. No. 5,296,375, issued Mar. 22, 1994 to Kricka et al. disclose microplatforms for detecting the presence of an analyte in a fluid.
U.S. Pat. No. 5,304,487, issued Apr. 19, 1994 to Kricka et al. disclose microplatforms for detecting the presence of an analyte in a fluid.
International Application, Publication No. WO94/16543, published on Jul. 21, 1994 in the name of Schutze et al., disclose the use of a laser optical trap for manipulating living cells.
U.S. Pat. No. 5,338,689 issued Aug. 16, 1994 to Yves et al; discloses a method for detecting antigens and antibodies.
U.S. Pat. No. 5,403,720 issued Apr. 4, 1995 to Sato et al. discloses a method for detecting microorganisms.
U.S. Pat. No. 5,427,946, issued Jun. 27, 1995 to Kricka et al. disclose microplatforms for detecting the presence of an analyte in a fluid.
U.S. Pat. No. 5,451,504 issued Sep. 19, 1995 to Fitzpatrick et al. discloses a membrane strip for detecting the presence of an analyte in a sample.
European Application, Publication No. EP634654, published on Oct. 4, 1995 in the name of Ventura disclose an apparatus for measuring purified water quality.
U.S. Pat. No. 5,460,940 issued Oct. 24, 1995 to Yves et al. discloses a method for detecting antigens and antibodies.
U.S. Pat. No. 5,460,979 issued Oct. 24, 1995 to Levine et al. discloses an indirect fluorescent assay of blood samples.
U.S. Pat. No. 5,491,067 issued Feb. 13, 1996 to Setcavage et al. discloses an agglutination reaction and separation vessel.
U.S. Pat. No. 5,496,697 issued Mar. 5, 1996 to Parce et al disclose an apparatus for detecting the effect of test compounds on cells.
U.S. Pat. No. 5,498,392, issued Mar. 12, 1996 to Kricka et al. disclose microplatforms for detecting the presence of an analyte in a fluid.
U.S. Pat. No. 5,506,141 issued Apr. 9, 1996 to Weinreb et al. discloses an apertured cell carrier.
U.S. Pat. No. 5,512,432 issued Apr. 30, 1996 to Lapierre et al. discloses methods for detecting antigens and antibodies.
U.S. Pat. No. 5,547,849 issued Aug. 20, 1996 to Baer et al. discloses an apparatus and method for volumetric capillary cytometry.
U.S. Pat. No. 5,556,764 issued Sep. 17, 1996 to Sizto et al. discloses an apparatus and method for cell counting and classification.
International Application, Publication No. WO96/12962, published on May 2, 1996 in the name of Biocircuits Corp. disclose detection of an analyte using particles and a specific binding pair in the presence of a transparent surface.
U.S. Pat. No. 5,637,469, issued Jun. 10, 1996 to Kricka et al. disclose microplatforms for detecting the presence of an analyte in a fluid.
U.S. Pat. No. 5,672,861 issued Sep. 30, 1997 to Fairley et al. discloses an automatic focusing device for a confocal laser microscope.
However, despite this cited prior art, there remains a need in the art for methods and apparatus to detect particulates in fluids, particularly cells in biological fluids, rapidly, simply, reliably and more economically than available using the prior art.
This invention provides an integrated, affinity-binding based, analytical apparatus for detecting particulates, particularly cells, suspended in a fluid, preferably a biological fluid. The invention provides a platform for performing an affinity-binding based assay for specifically binding particulates such as cells, preferably microbial cells, especially bacterial cells, and mammalian cells, especially hematopoietic cells, and a detection means for detecting the particulates specifically bound to a defined surface or chamber comprising the platform. Methods for using the platforms of the invention are also provided.
In one aspect of the invention is provided an affinity-binding based, analytical apparatus for detecting particulates suspended in a fluid. The apparatus provided by the invention comprises a platform having a surface defining a detection chamber, whereby a specific binding reagent is deposited on the surface of the chamber and specifically binds the particulate to be detected. In preferred embodiments, the specific binding reagent is an antibody, a ligand, a lectin, an integrin, an antigen, a receptor, a carbohydrate or an adhesion molecule. Preferably, the surface of the detection chamber is also treated with a blocking compound that discourages non-specific binding to the surface of the chamber. In another preferred embodiment, the platform is a rotatable structure, most preferably a disk. In a preferred embodiment of this aspect of the invention, the disk is a microplatform as disclosed in co-owned and co-pending Ser. No. 08/768,990, filed Dec. 18, 1996, incorporated by reference. The platforms of the invention also preferably comprise fluid sample input means, overflow reservoirs, wash buffer reservoirs, and fluid waste receptacles, in fluid connection with each other as described herein, as well as air displacement vents or orifices, or means for removing the fluid component of a sample applied thereto.
Means for detecting specifically-bound particles in the surface or chamber are also provided. Preferred embodiments of detecting means are a light source, particularly a monochromatic light source, and a detector therefor. In addition, preferred embodiments of the platforms of the invention comprise reservoirs containing detectable labeling reagents and moieties for detecting particulates retained on the detection chambers of the platforms. In preferred embodiments, said reagents and moieties comprise stains, preferably histochemical stains and most preferably vital stains, that specifically bind to the particulates, most preferably cells, in the detection chambers of the platforms of the invention. In additional preferred embodiments, said reagents and moieties comprise immunochemical reagents, preferably antisera and antibodies and most preferably monoclonal antibodies, that specifically bind to the particulates, most preferably cells, in the detection chambers of the platforms of the invention. In preferred embodiments, said antisera and antibodies are labeled with a detectable label. Preferred detectable labels include fluorescent labels and enzymatic moieties capable of converting a substrate to a detectable product. In alternative preferred embodiments, the detectable reagents comprise a first antisera or antibody specific for the particulate to be detected, most preferably a cell, and a second antisera or antibody that specifically recognizes and binds to said first antisera or antibody, and is itself detectably labeled. Preferred detectors include photodetectors, most preferably photodiodes, avalanche photodiodes, photocells and photomultiplier tubes.
In a second embodiment of this aspect of the invention is provided an apparatus that comprises a platform having a surface defining a cell accumulation chamber, whereby particulates that are cells, preferably microbial cells, especially bacterial cells, and mammalian cells, especially hematopoietic cells, accumulate in the chamber and are detected therein. Preferably, in certain embodiments, the chamber also comprises a filtering means having a pore size that prevents the cells from leaving the chamber when the fluid comprising the sample is replaced by buffer solutions, detection reagents or other fluid volumes. In other embodiments, the chamber preferably comprises a non-specific cell adhesion coating on the surface thereof that retains the cells in the chamber. In another preferred embodiments, the surface is treated to permit the cells to attach and multiply in the cell accumulation chamber of the platforms of the invention.
In additional preferred embodiments, the platform is a rotatable structure, most preferably a disk. In a preferred embodiment of this aspect of the invention, the disk is a microplatform as disclosed in co-owned and co-pending Ser. No. 08/768,990, filed Dec. 18, 1996, incorporated by reference. The platforms of the invention also preferably comprise fluid sample input means, overflow reservoirs, wash buffer reservoirs, fluid waste receptacles, or reservoirs containing an amount of a detectable labeling moiety for labeling the cells retained in the accumulation chamber, in fluid communication with each other as described herein, or air displacement vents or orifices, or means for removing the fluid component of a sample applied thereto.
In alternative embodiments, the platforms of the invention are provided to detect, quantitate and characterize the effect(s) of a test compound on a cell, most preferably on the metabolism, physiology or viability of the cell. In such embodiments, platforms are provided with reservoirs containing a test compound and other components therefor. In such embodiments, cells retained in the cell accumulation chamber of a platform of the invention are treated with a test compound contained in a reservoir in fluid communication with the cell accumulation chamber. Said test compound is transferred to the cell accumulation chamber, most preferably replacing the fluid sample, for a time and under conditions wherein the test compound can have an effect on the cell. Alternatively, the test compound can comprise a component of the cell accumulation chamber as provided. Detection of cell viability, for example using vital stains, or cellular physiology or metabolism by detecting metabolites or other cell products produced in response to the test compound is achieved using the platforms of the invention, wherein reagents for detecting said effect-associated molecules produced by the cells are introduced into the cell accumulation chamber prior to detection. In these embodiments, detection of the effect-associated molecules is achieved using reagents specific for said molecules and detection means specific for said reagents. Most preferably, the effect-associated molecules are detected using photodetectable reagents such as dyes, most preferably fluorescent dyes, which are contained in a reservoir in fluid communication with the cell accumulation chamber and delivered thereto after treatment of the cells with the test compound.
Means for detecting specifically-bound particles in the surface or chamber are also provided. Preferred embodiments of detecting means are a light source, particularly a monochromatic light source, and a detector therefor. In addition, preferred embodiments of the platforms of the invention comprise reservoirs containing detectable labeling reagents and moieties for detecting particulates retained on the cell accumulation chambers of the platforms. In preferred embodiments, said reagents and moieties comprise stains, preferably histochemical stains and most preferably vital stains, that specifically bind to the particulates, most preferably cells, in the cell accumulation chambers of the platforms of the invention. In additional preferred embodiments, said reagents and moieties comprise immunochemical reagents, preferably antisera and antibodies and most preferably monoclonal antibodies, that specifically bind to the particulates, most preferably cells, in the cell accumulation chambers of the platforms of the invention. In preferred embodiments, said antisera and antibodies are labeled with a detectable label. Preferred detectable labels include fluorescent labels and enzymatic moieties capable of converting a substrate to a detectable product. In alternative preferred embodiments, the detectable reagents comprise a first antisera or antibody specific for the particulate to be detected, most preferably a cell, and a second antisera or antibody that specifically recognizes and binds to said first antisera or antibody, and is itself detectably labeled. Preferred detectors include photodetectors, most preferably photodiodes, avalanche photodiodes, photocells and photomultiplier tubes.
Additional embodiments of each of these aspects of the invention include platforms comprising a multiplicity of the components of the cell detection arrays of the invention as defined herein, thereby providing for the analysis of multiple aliquots of the same sample or multiple samples on the same platform.
In a second aspect of the invention, an affinity-binding based, analytical apparatus is provided that is a combination of two elements. The first element is a platform as described herein. In preferred embodiments of these aspects of the invention, the platform is a rotatable platform having a means for being rotated about a central axis comprising a rotational element, preferably a hole for a spindle. In these aspects of the invention various components of the platform are connected to one another by channels, most preferably microchannels as defined herein. The second element in this aspect of the invention is a device comprising a holding means for accommodating the platforms of the invention, most preferably also including detecting means for detecting particulates and most preferably cells on the platforms of the invention. In preferred embodiments, the devices of the invention are provided as a device that comprises rotating means and controlling means thereof, and components of a detecting means operably positioned to detect binding of particulates on the platform surface and most preferably in a detection or cell accumulation chamber of the platform. In these aspects of the invention, fluid displacement through the components of the platforms of the invention is motivated by centripetal force produced by rotation of the platform about the central axis at a speed and for a time determined by controlling means comprising the device. In a preferred embodiment, the platform and device comprise a disk and player/reader device as disclosed in co-owned and co-pending U.S. Ser. No. 08/768,990, filed Dec. 18, 1996 and incorporated by reference.
Methods for analyzing a fluid, preferably a biological fluid, to detect particulates suspended therein using the platforms of the invention are also provided. In preferred embodiments, the methods provided by the invention comprise the steps of, first, applying a fluid sample to the surface of the platform, preferably to a fluid sample input means, and most preferably wherein said means further comprises means for metering a specific volume of the fluid into a detection or cell accumulation chamber on the surface of the platform.
In certain embodiments of the methods of the invention a metered amount of the fluid sample applied to the platform is transferred to a detection or cell accumulation-chamber. For the purposes of this invention, the term xe2x80x9ca metered amountxe2x80x9d will be understood to mean a volumetric amount of It the fluid sample that fills a metering means in the fluid sample input means, wherein volumetric amounts greater than the metered amount are removed from the fluid sample input means into an overflow chamber or fluid waste receptacle. In preferred embodiments, the metered amount of the fluid sample provided on an inventive platform is from about 10 xcexcL to about 500 xcexcL.
In certain embodiments of the methods of the invention the amount of the fluid sample applied to the platform is transferred to a detection chamber coated with a specific binding reagent and incubated thereupon for a time and under conditions wherein specific binding is achieved between the particulates comprising the fluid sample and the specific binding reagent, thereby immobilizing the particulate in the detection chamber, and removing the fluid sample from the chamber. In preferred embodiments, the cells in the detection chamber are washed with a solution, preferably a buffer and more preferably a buffer comprising a component, preferably a salt or detergent, that dissociates particulates retained in the chamber by non-specific binding unrelated to binding of the particulate to the specific binding reagent and does not dissociate binding of the particulate to the specific binding reagent; said washing solution is preferably removed from the chamber prior to cell detection to effect removal of non-specifically retained particulates. The selective removal of non-specifically bound particulates is accomplished by precisely controlling the surface shear force exerted on the particulates by the fluid flowing through the detection chamber. Thereafter the presence, identity and number of particulates in the detection chamber are detected. In certain embodiments of the methods of the invention, a solution containing a reagent for detecting a particulate in the detection chamber is added to the chamber before detection of the particulate is accomplished, most preferably by contacting the cells retained in the detection chamber with such reagents. In certain embodiments of this aspect of the methods of the invention, particulates, preferably cells are stained with a specific dye either prior to application to the platform or after the particulates are retained in the detection chamber. In additional preferred embodiments, the reagent is an antisera or antibody, most preferably a monoclonal antibody, linked to a detectable marker such as a fluorescent compound. In other preferred embodiments, the reagent is an antisera or antibody, most preferably a monoclonal antibody, linked to an enzyme capable of converting a substrate to a detectable product; in such embodiments, the appropriate substrate is also added to the particulates prior to application to the platform or more preferably after the particulates are retained in the detection chamber, in a concentration, for a time and under conditions whereby the substrate is converted to the detectable product. In preferred embodiments, the particulates detected in fluid samples using the methods of the invention are cells, preferably microbial cells, especially bacterial cells, and mammalian cells, most preferably hematopoietic cells.
In other embodiments of the methods of the invention, the metered amount of the fluid sample applied to the platform is transferred to a cell accumulation chamber that retains the cells therein upon evacuation of the chamber of the fluid sample or replacement of the fluid sample with other fluid components of the platforms of the invention. In these embodiments, the fluid sample is incubated in the cell accumulation chamber for a time and under conditions wherein cells are retained in the chamber, after which the fluid sample is removed from the chamber. In preferred embodiments, the cells in the cell accumulation chamber are washed with a solution, preferably a buffer and more preferably a buffer comprising a component, preferably a salt or detergent, that dissociates non-cellular particulates from the chamber but does not remove the cells from the chamber; said washing solution is preferably removed from the chamber prior to cell detection to effect removal of non-cellular particulates. Thereafter the presence, identity and number of cells in the accumulation chamber are detected.
In alternative embodiments, the effect of a test compound on a cell, most preferably on the metabolism, physiology or viability of the cell, is determined using the methods of the invention. In such embodiments, cells retained in the cell accumulation chamber of a platform of the invention are treated with a test compound for a time and under conditions wherein the test compound can have an effect on the cell. Detection of cell viability, for example using vital stains, or cellular physiology or metabolism by detecting metabolites or other cell products is achieved using the platforms of the invention, wherein reagents for detecting said effect-associated molecules produced by the cells are introduced into the cell accumulation chamber prior to detection. In these embodiments, detection of the effect-associated molecules is achieved using reagents specific for said molecules and detection means specific for said reagents. Most preferably, the effect-associated molecules are detected using photodetectable reagents such as dyes, most preferably fluorescent dyes.
In certain embodiments of the methods of the invention, a solution containing a reagent for detecting a particulate in the cell accumulation chamber is added to the chamber before detection of the particulate is accomplished, most preferably by contacting the cells retained in the chamber with such reagents. In certain embodiments of this aspect of the methods of the invention, particulates, preferably cells are stained with a specific dye either prior to application to the platform or after the particulates are retained in the cell accumulation chamber. In additional preferred embodiments, the reagent is an antisera or antibody, most preferably a monoclonal antibody, linked to a detectable marker such as a fluorescent compound. In other preferred embodiments, the reagent is an antisera or antibody, most preferably a monoclonal antibody, linked to an enzyme capable of converting a substrate to a detectable product; in such embodiments, the appropriate substrate is also added to the particulates prior to application to the platform or more preferably after the particulates are retained in the cell accumulation chamber, in a concentration, for a time and under conditions whereby the substrate is converted to the detectable product. In preferred embodiments, the particulates detected in fluid samples using the methods of the invention are cells, preferably microbial cells, especially bacterial cells, and mammalian cells, most preferably hematopoietic cells.
Certain preferred embodiments of the apparatus of the invention are described in greater detail in the following sections of this application and in the drawings.